Genotypic and Phenotypic Characterization of Replication-Competent HIV-2 Isolated from Controllers and Progressors.

Although some individuals with HIV-2 develop severe immunodeficiency and AIDS-related complications, most may never progress to AIDS. Replication-competent HIV-2 isolated from asymptomatic long-term non-progressors (controllers) have lower replication rates than viruses from individuals who progress to AIDS (progressors). To investigate potential retroviral factors that correlate with disease progression in HIV-2, we sequenced the near full-length genomes of replication-competent viruses previously outgrown from controllers and progressors and used phylogeny to seek genotypic correlates of disease progression. We validated the integrity of all open reading frames and used cell-based assays to study the retroviral transcriptional activity of the long terminal repeats (LTRs) and Tat proteins of HIV-2 from controllers and progressors. Overall, we did not identify genotypic defects that may contribute to HIV-2 non-progression. Tat-induced, LTR-mediated transcription was comparable between viruses from controllers and progressors. Our results were obtained from a small number of participants and should be interpreted accordingly. Overall, they suggest that progression may be determined before or during integration of HIV-2.

HIV-1 Resistance Dynamics in Patients With Virologic Failure to Dolutegravir Maintenance Monotherapy.

Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.

Characterization of recombinant influenza A virus as a vector for HIV-1 p17Gag.

We have generated a recombinant influenza A virus with the HIV-1 p17(Gag) (rFlu-p17) gene inserted into the influenza virus neuraminidase (NA) gene. Expression of HIV-1 p17 protein was detected by conventional Western blot analysis and also by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of rFlu-p17 infected cells. The latter method does not depend on protein-specific antibody preparations and proved to be a powerful tool to detect proteins of interest. Next, antigen presentation of p17 expressed after infection of antigen-presenting cells was determined. Cloned p17-specific CD8+ T-cells were co-cultured with rFlu-p17 infected B-cells and produced IFN-gamma upon stimulation. Furthermore, we showed that immunization with rFlu-p17 elicited a humoral immune response in mice. This study shows that replication-deficient rFlu-p17 is antigenic in vitro and immunogenic in vivo and warrants further development as a candidate vaccine vector.

CCR5-restricted HIV type 2 variants from long-term aviremic individuals are less sensitive to inhibition by beta-chemokines than low pathogenic HIV type 1 variants.

Many HIV-2-infected individuals maintain low, often undetectable, viral loads for prolonged periods. Virus and/or host factors that contribute to this high level of virus control are largely unknown. Previously we demonstrated that HIV-2 variants from long-term aviremic individuals have relatively low replication kinetics in vitro in comparison to HIV-1 variants. We hypothesized that the relatively low replication rates of HIV-2 in vitro as well as the high level of virus control in vivo might be explained by HIV-2 replication being more sensitive to inhibitory host factors like beta-chemokines or other CD8+ T cell-derived factors than HIV-1 replication. To test this we determined the effect of exogenously added beta-chemokines and healthy donor CD8+ T cells on the in vitro virus production of HIV-2 and HIV-1 variants from long-term nonprogressors (LTNPs). Contrary to expectations, HIV-2 replication was inhibited less efficiently by RANTES and MIP-1alpha than HIV-1 replication. CD8+ T cells from 8 of 12 healthy donors reduced HIV replication minimally 2-fold. Interestingly, cells from five of these donors inhibited HIV-1 but hardly affected HIV-2 replication, while the reverse was observed for cells from one donor. For HIV-1, but not HIV-2, the magnitude of the antiviral effect of CD8+ T cells correlated with their effect on RANTES levels in culture supernatants. Our findings indicate that RANTES is a more important factor of CD8+ T cell-associated anti-HIV-1 activity than it is of HIV-2 activity and that the benign clinical course of HIV-2 infection is not due to enhanced beta-chemokine sensitivity of HIV-2 variants.

In vitro replication capacity of HIV-2 variants from long-term aviremic individuals.

To establish whether efficient suppression of virus replication in HIV-2-infected individuals is associated with low replicative capacity of HIV-2, replication kinetics of HIV-2 variants from long-term aviremic individuals was analyzed and compared with that of the relatively slow-replicating HIV-1 variants from asymptomatics and long-term nonprogressors (AS/LTNP). On average, HIV-2 from aviremic individuals had lower replication rates than HIV-1 variants from AS/LTNP in cells of 8 donors (0.45 log10 [range 0.14-0.77] vs. 0.58 log10 [range 0.32-0.99] pg RT/ml/day, P = 0.036). The relatively low replication rate of HIV-2 compared to HIV-1 variants was not related to different sensitivities to inhibition by CD8+ T cells or different degrees of infectivity. HIV-2 replication rates increased with progressive infection and with switch from CCR5 to CXCR4 usage. The relatively low replicative capacity of HIV-2 variants from aviremic individuals likely contributes to the low viral load and benign course of infection in these individuals.

HIV-2-infected individuals with undetectable plasma viremia carry replication-competent virus in peripheral blood lymphocytes.

Using an optimized HIV co-culture protocol it was possible to isolate infectious HIV-2 variants from 6 HIV-2-infected individuals who had undetectable plasma viremia and maintained high CD4 T-cell numbers for prolonged periods. This shows for the first time that HIV-2-infected individuals with no demonstrable in vivo virus production carry replication-competent virus in peripheral blood mononuclear cells (PBMCs). The frequency of PBMCs with infectious virus was low, ranging from 0.01-0.9 infectious units per million (IUPM) CD4 T cells with a median value of 0.2 IUPM. In comparison, viremic HIV-2-infected individuals had a 2-log higher median infectious load (36 IUPM, range 1-673; P = 0.003). HIV-2 infectious load correlated with CD4 counts (rs = -0.88, P < 0.0001). The low infectious load in aviremic HIV-2-infected persons is reminiscent of what has been observed for HIV-1 infection controlled by highly active antiretroviral therapy.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes.

Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Antibody-mediated enhancement of human immunodeficiency virus type 1 infectivity is determined by the structure of gp120 and depends on modulation of the gp120-CCR5 interaction.

In this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different phenotypes. We found that the V3 region was the main determinant of antibody-mediated enhancement and coreceptor specificity but that the overall structure of gp120 was also important for these properties. Constructs susceptible to antibody-mediated enhancement preferentially use CCR5 as a coreceptor, in contrast to constructs that were neutralized or not affected. Using monoclonal antibodies directed against CD4 or CCR5, we were able to show that antibody-mediated enhancement was CD4 dependent. Altogether, our results suggest that the modulation of the interaction of gp120 with CCR5 is the mechanism underlying antibody-mediated enhancement of HIV-1 infectivity.

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.